This is a repository copy of Extracellular vesicle microRNA cargo is correlated with HPV status in oropharyngeal carcinoma. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/140646/ Version: Accepted Version Article: Peacock, B., Rigby, A., Bradford, J. orcid.org/0000-0002-7771-914X et al. (4 more authors) (2018) Extracellular vesicle microRNA cargo is correlated with HPV status in oropharyngeal carcinoma. Journal of Oral Pathology and Medicine, 47 (10). pp. 954-963. ISSN 0904-2512 https://doi.org/10.1111/jop.12781 This is the peer reviewed version of the following article: Peacock B, Rigby A, Bradford J, et al. Extracellular vesicle microRNA cargo is correlated with HPV status in oropharyngeal carcinoma. J Oral Pathol Med. 2018;47:954–963, which has been published in final form at https://doi.org/10.1111/jop.12781. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. [email protected]https://eprints.whiterose.ac.uk/ Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request.
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This is a repository copy of Extracellular vesicle microRNA cargo is correlated with HPV status in oropharyngeal carcinoma.
White Rose Research Online URL for this paper:http://eprints.whiterose.ac.uk/140646/
Version: Accepted Version
Article:
Peacock, B., Rigby, A., Bradford, J. orcid.org/0000-0002-7771-914X et al. (4 more authors)(2018) Extracellular vesicle microRNA cargo is correlated with HPV status in oropharyngeal carcinoma. Journal of Oral Pathology and Medicine, 47 (10). pp. 954-963. ISSN 0904-2512
https://doi.org/10.1111/jop.12781
This is the peer reviewed version of the following article: Peacock B, Rigby A, Bradford J, et al. Extracellular vesicle microRNA cargo is correlated with HPV status in oropharyngeal carcinoma. J Oral Pathol Med. 2018;47:954–963, which has been published in final form athttps://doi.org/10.1111/jop.12781. This article may be used for non-commercial purposes inaccordance with Wiley Terms and Conditions for Use of Self-Archived Versions.
Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item.
Takedown
If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request.
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jop.12781 This article is protected by copyright. All rights reserved.
DR STUART HUNT (Orcid ID : 0000-0001-5572-1030)
Article type : Original Article
Extracellular vesicle microRNA cargo is correlated with HPV-status in oropharyngeal carcinoma
Running title: EV miRNA cargo in OPSCC
Ben Peacock1, Alice Rigby1, James Bradford2,4, Ryan Pink3, Keith Hunter1, Daniel Lambert1,4 and
Stuart Hunt1
1The School of Clinical Dentistry, The University of Sheffield, UK; 2Department of Oncology and
Metabolism, Medical School, The University of Sheffield, UK; 3Department of Biological and
Medical Sciences, Oxford Brookes University, UK; 4Sheffield Institute for Nucleic Acids,
University of Sheffield, UK
Corresponding author: Dr Stuart Hunt, Unit of Oral and Maxillofacial Medicine, Surgery and
Pathology, The School of Clinical Dentistry, The University of Sheffield, 19 Claremont Crescent,
This article is protected by copyright. All rights reserved.
Keywords: Extracellular vesicles, microRNA, oropharyngeal carcinoma, human papillomavirus,
head and neck cancer
Abstract
Background: The incidence of human papilloma virus positive (HPV+) oropharyngeal
squamous cell carcinoma (OPSCC) has increased rapidly in recent decades. These tumours have
a favourable outcome compared to HPV-negative (HPV-) OPSCC. However, HPV+ tumours are
more likely to metastasise to distant sites, suggesting a difference in how these tumour subtypes
interact with the metastatic niche. Extracellular vesicles (EVs) have emerged as important
players in cell-to-cell communication and are a potential source of biomarkers for cancer
diagnosis. This study aims to characterise the microRNA cargo of small EVs released by HPV+
and HPV- OPSCC cell lines.
Methods: EVs produced by HPV+ (SCC2 and SCC90) and HPV- (SCC72 an SCC89) OPSCC cells
were characterised by tunable resistive pulse sensing (TRPS) and western blotting. RNA was
extracted from EVs and analysed by small RNA sequencing. A bioinformatics approach was used
to identify EV miRNA signatures associated with HPV status.
Results: HPV- OPSCC cells produced more EVs than HPV+ OPSCC cells. EVs were positive for the
common EV markers CD63, CD9 and TSG101. Unbiased hierarchical clustering analysis of EV
miRNA cargo revealed that samples clustered based on HPV status. 14 miRNA were enriched in
HPV+ cell-derived EVs, whereas, 19 miRNA were enriched in EVs derived from HPV- cell lines.
Conclusions: Here we identify EV miRNA signatures indicative of the HPV status of the parent
cell. This may provide a platform from which to validate salivary or blood-based biomarkers
with utility for early detection and stratifying risk in OPSCC patients.
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Introduction
The incidence of human papillomavirus-positive (HPV+) oropharyngeal squamous cell
carcinoma (OPSCC) has risen dramatically in recent decades. This is in contrast to HPV-negative
(HPV-) OPSCC, which continues to decline.1,2 It has been predicted that by 2020 the annual
incidence of HPV+ OPSCC will overtake the number of cervical cancers and will represent the
majority of head and neck cancers by 2030.1 HPV+ OPSCC have been shown to have a more
favourable outcome due to an increased sensitivity to radiotherapy and lower risk of recurrence
at the primary site when compared to HPV- OPSCC.3 However, some patients with HPV+ OPSCC
have poor outcomes due to formation of distant metastases, which typically manifest later than
in HPV- patients, can occur at unusual sites and may display a disseminating or explosive
phenotype.4
Extracellular vesicles (EVs) are nanoscale, membrane enclosed vesicles released by cells to
facilitate cell-to-cell communication.5 EVs contain molecular cargo such as nucleic acids and
protein.6 They are present in all biological fluids7, have attracted much attention as a source of
biomarkers for disease diagnosis and also as novel therapeutic targets, as their contents appear
to be altered in disease.8
There is evidence that HPV alters the expression of host cell microRNAs9-11 and the cargo of EVs
released by cervical cancer cells.12,13 However, despite the common viral aetiology there are no
similar studies in OPSCC. To address this gap in the literature, we characterised the small EVs
released by HPV+ and HPV- OPSCC cell lines and performed small RNA sequencing on EV cargo
to elucidate the miRNA signature associated with these different tumours.
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Methods
Cell culture ‒ During this study four oropharyngeal carcinoma cell lines were used, which were
received under Material Transfer Agreement from Prof. S. Gollin, University of Pittsburgh School
of Public Health, Pittsburgh, PA: SCC2 (hypopharynx), SCC90 (base of tongue), SCC72 (tonsil)
and SCC89 (tonsil). Two were HPV16 positive (SCC2 and SCC90) and two were HPV negative
(SCC72 and SCC90). HPV status was confirmed by cytology testing carried out by the Directorate
of Laboratory Medicine at The Royal Hallamshire Hospital, Sheffield Teaching Hospitals NHS
Foundation Trust, Sheffield. Cell lines were routinely cultured in Dulbecco╆s Modified Eagle╆s Medium (DMEM) supplemented with 10% (v/v) FBS and 2 mM L-glutamine (all from Sigma-
Aldrich, Dorset, UK). Cells were maintained at 37°C, 5% CO2 in a humidified environment.
Conditioned medium and EV isolation - Cells were seeded in complete growth medium and
allowed to adhere overnight. Monolayers were washed with PBS and the medium exchanged for
DMEM supplemented with 5% (v/v) EV-depleted FBS (centrifuged at 100,000 x g for 18 hours
and filtered through a 0.2 µm syringe filter) and incubated for 24-72 h. Conditioned medium
was centrifuged at 300 x g for 10 min, 2000 x g for 10 min, 10,000 x g for 30 min. The
supernatant was concentrated to 0.5 ml using a Vivaspin-20 (100 MWCO) device (GE
Healthcare, Buckinghamshire, UK). EVs were separated from soluble factors by size exclusion
chromatography using Sepharose CL-2B (GE Healthcare, Uppsala, Sweden) stacked in
disposable Econo-Pac columns (Biorad, Watford, UK) and eluted in PBS. Where required, EVs
were pelleted by ultracentrifugation at 100,000 x g for 1 h.
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Tunable resistive pulse sensing (TRPS) - EVs were analysed by TRPS using a qNano instrument
(iZON, Oxford, UK). An NP100 nanopore was selected to measure small EVs. Typical settings
were 45 mm stretch, 0.5 V, 10 mbar pressure, but small alterations were made as required to
maintain a current of ~125 nA.
Western blotting - Cell and EV pellets were resuspended in RIPA buffer supplemented with
protease inhibitor cocktail. Protein concentration was calculated by BCA assay (Thermo
Fisher,Waltham, USA) and 20 µg of protein per sample was separated by SDS-PAGE on 10%
polyacrylamide gels. Proteins were transferred to nitrocellulose membrane using the BioRad
Turbo Blot system. Membranes were blocked for 1 h at room temperature in 5% (w/v)
skimmed milk powder in TBST. Primary antibodies were added at the following dilutions at 4°C
Small RNA sequencing - EV pellets were resuspended in lysis buffer from the miRCURY RNA
isolation kit (Exiqon, Vedbaek, Denmark) and the protocol was followed according to manufacturer╆s instructions┸ including the optional on-column DNase treatment. Small RNA
sequencing was performed at the Clinical Research Facility, University of Edinburgh: RNA
quality and integrity was determined by Agilent 2100 Bioanalyzer Instrument with the RNA
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6000 Pico Kit. RNA concentration was quantified using the Qubit 2.0 fluorometer and the Qubit
RNA HS Assay Kit. Library preparation was performed using the Ion Total RNA-Seq Kit v2. RNA
was hybridised and ligated before being reverse transcribed to cDNA and purified using
magnetic beads. cDNA was amplified with Ion Torrent adapters and barcodes for 18 cycles of
PCR before being purified. Library yields were quantified using the Qubit 2.0 fluorometer and
the dsDNA HS Kit. Libraries were combined in equimolar amounts for template preparation
before sequencing on the Ion Proton Instrument on a P1 v3 chip. Output files were converted to
BAM files by an automated workflow using the Torrent Suite software v5.0.4. In addition, the
smallRNA_analysis plug-in v5.0.3.0 was used to analyse microRNA reads. Unmapped reads were
aligned to the whole genome to rescue miRBase unaligned reads.
Quantitative real-time PCR - Cellular and EV RNA was reverse transcribed using the small RNA
reverse transcription kit with the appropriate RT primer (Applied biosystems, California, USA).
QPCR reactions consisted of Taqman mastermix, the appropriate primer/probe sets (Applied
biosystems, California, USA) and cDNA from specific miRNA RT. QPCR was performed using a
Rotor Gene Q thermal cycler (Qiagen, Hilden, Germany). Data was normalised to hsa-let-7a-5p
(a miRNA expressed at constant levels according to our data) and expressed relative to the
sample with lowest abundance【expression using the つつCt method┻
Processing of small RNA sequencing data ‒ Raw sequence reads were aligned to miRBase using
the smallRNA_analysis plug-in v5.0.3.0 within Torrent Suite software v5.0.4. Any unmapped
reads were further aligned to the whole genome to rescue miRBase unaligned reads. Raw
counts across each miRNA were then input to a differential expression (DE) analysis workflow
through the web app developed by the bioinformatics core, Center for Research Informatics
(CRI), University of Chicago (https://gallery.shinyapps.io/DEApp/). Briefly, the four cell lines
were grouped by HPV status and the raw counts normalized. An exclusion criteria of <50 reads
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per Million (RPM) in at least 2 columns/cell lines reduced the number of miRNAs analysed from
575 to 233. DE analysis and pathway enrichments was then performed on raw count data from
the two groups using DESeq214 with a cutoff for log2 Fold Change (log2 FC) of at least 1.5 and a
P value <0.05.
Pathway analysis - Experimentally validated miRNA targets were determined using miRTarBase
v.815 for HPV-/+ EV enriched sets. Transcripts targeted by multiple miRNA were analysed for
pathway enrichment using DAVID16,17 to identify head and neck cancer-associated pathways.
Stringency criteria were applied using Benjamini-Hochberg correction <0.05.
Results
HPV negative OPSCC cell lines produce more EVs
Prior to purification of OPSCC-derived EVs, we sought to characterise the size profile of small
EVs present in conditioned medium. TRPS using a qNano instrument revealed numerous small
particles, most likely EVs, between 50 to 150 nm in diameter (Fig 1). Conditioned medium
derived from one of the HPV- cell lines, SCC89, contained EVs with a larger modal diameter (Fig
1A) and a more normal size distribution. Conditioned medium from the other cells examined,
both HPV+ and HPV-, contained EVs with a skewed normal distribution (Fig 1C). Normalisation
of EV concentration in conditioned medium to the number of producing cells revealed that HPV-
cells produced up to two fold more than HPV+ cells (P value <0.05) (Fig 1B).
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Isolation of small EVs from conditioned medium
Small EVs were isolated from the conditioned medium of OPSCC cell lines by a combined
differential centrifugation and size exclusion chromatography (SEC) approach. EV-rich fractions
were combined and EVs were pelleted by ultracentrifugation (100,000 x g for 1 h). Pelleted EVs
were resuspended in 50 µl PBS or protein lysis buffer for subsequent TRPS or western blot
analysis, respectively. TRPS confirmed the isolation of small EVs with similar modal diameters
to the particles detected in conditioned medium (compare Fig 1A with Fig 2A). Analysis of the
EV size profiles showed that all samples contained particles between 50 to 150 nm in diameter
with a skewed normal distribution (Fig 2C). Interestingly, the size profile of purified SCC89 EVs
was similar to the other samples, suggesting that a proportion of the EVs larger than 80 nm
(present in conditioned medium samples) were eliminated during the purification procedure
(Fig 2C). Western blot analysis revealed that EV lysates were positive for the EV markers CD63,
CD9 or TSG101. Whereas, the Golgi marker GM130 was only detected in cell lysates (Fig 2B).
EVs isolated from OPSCC cell lines contain a variety of small RNA molecules
RNA was extracted from EV pellets and subjected to Bioanalyzer analysis, which showed that
samples were enriched with small RNA species <200 nt in length (Fig 3A-D). Small RNA
sequencing produced 8-13 million reads per sample, with 89-94% mapped to mirBase and the
human genome. Of these mapped reads, 2-15% aligned to miRNA sequences. Other RNA species
that were identified included mRNA, rRNA, tRNA, snoRNA, snRNA and lincRNA (Fig 3E). Raw
counts were converted to reads per million (RPM). For each sample 343-443 unique miRNA
were identified. 63-82 miRNA had high abundance (>1000 RPM), 73-92 miRNA had
intermediate abundance (100-1000 RPM) and 117-133 miRNA had low abundance (10-100
RPM) (Fig 3F).
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EV miRNA profile correlates with HPV status
MiRNA data was converted to log2 RPM and unbiased hierarchical clustering analysis revealed
that the global EV miRNA signature correlated with HPV status, with SCC2 and SCC90 (HPV+)
and SCC72 and SCC89 (HPV-) clustering together (Fig 3G). In order to identify specific miRNA
associated with HPV status, fold difference was calculated between HPV+ and HPV- groups using
DEseq2, with a cutoff of 1.5 log2 fold change and P value of <0.05. 14 miRNA were identified
that were enriched in EVs from HPV+ cells and 19 miRNA were identified that were enriched in
EVs from HPV- cells (Table 1). In addition, 9 miRNA were very abundant in all EV samples
(>5000 RPM), independent of HPV status (Table 1). We next sought to validate small RNA
sequencing data by quantitative real-time PCR (QPCR). miR-29b-3p and miR-20b-5p were
chosen as miRNA that were enriched in EVs derived from HPV- or HPV+ cells, respectively. The
pattern of enrichment seen in small RNA sequencing data (Fig 4A,D) was also observed by QPCR
analysis (Fig 4B,E). We also examined the cellular expression level of each miRNA, which
showed that the EV abundance showed a similar trend to that of cellular expression (Fig 4C,F).
Pathway analysis
Due to certain miRNA being enriched in either EVs derived from HPV+ or HPV- cells, we used a
bioinformatics approach to determine which cell signalling pathways in the tumour
microenvironment might be affected by cellular uptake of EVs released by cancer cells in OPSCC.
In addition, we wanted to know if the analysis could elucidate which pathways were being
dysregulated in cancer cells (by ejection of miRNA via EVs). We used miRTarBase to identify
experimentally validated targets for each miRNA in our HPV+/- enriched lists and used these
transcripts in conjunction with DAVID to determine which signalling pathways would most
likely be affected by the uptake or release of EV miRNA cargo. A number of overlapping
pathways were shown to be targeted by miRNA enriched in EVs from HPV+ and HPV- cells
including PI3K-Akt, FoxO, HIF-1, mTOR and p53 signalling pathways (Table 2).
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Discussion
In the current study, we show for the first time the miRNA signature associated with EVs
released by HPV+ and HPV- OPSCC cells cultured in vitro. A number of miRNA were identified
that were either enriched in HPV- or HPV16+ cell derived EVs. We also identified miRNA that
were highly abundant in all EVs, independent of HPV status of the producing cell. We found
miRNA that were abundant in EVs from HPV+ OPSCC cells that had previously been reported as
cargo of EVs from HPV18+ HeLa cervical carcinoma cells, including miR-99a-5p, miR-27a-3p and
miR-27b-3p.13 Our data indicate that miR-99a-5p is enriched in EVs derived from HPV+ OPSCC.
Whereas, miR-27a-3p and miR-27b-3p are abundant in EVs produced by HPV+ and HPV- cells.
The most highly enriched miRNA in HPV+ EVs was miR-363-3p, which has been shown to be the
most differentially expressed miRNA when comparing HPV+ and HPV- tonsillar squamous cell
carcinoma ex vivo tissue sections.18 Two other members of the miR-106-363 cluster were
enriched in HPV+ OPSCC-derived EVs, miR-106a-5p and miR-20b-5p/miR-20b-3p. miR-106a-5p
and miR-20b-5p have recently been shown to be upregulated in breast cancer tissues and
enriched in circulating serum EVs of breast cancer patients.19 miR-20b-5p has been described as
a core HPV upregulated miRNA because it is consistently upregulated in HPV+ OPSCC20 and
cervical cancer tissues.18 Increased miR-20b-5p expression in cervical cancer is dependent on
the viral E6 oncoprotein and has been shown to promote cancer cell invasion.21 However,
expression of the normally silent miR-20b-5p and other miRNA cluster members (miR-106a-5p
and miR-363-3p) caused a decrease in proliferation in oral squamous cell carcinoma cells 22,
highlighting the cancer-specific role of certain miRNAs. We found an enrichment of miR-9-5p
and miR-9-3p in HPV+ EVs, which is consistent with reports of their overexpression in HPV+
OPSCC20 and HPV+ cervical cancer.23
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The most highly enriched miRNA in HPV- EVs was miR-767-5p, which has been shown to be
overexpressed in some tumours and to target members of the TET family of tumour
suppressors.24 miR-105-5p was similarly enriched in HPV- EVs and has been reported to
promote breast cancer metastasis by EV mediated targeting of endothelial cells.25 We found that
miR-100-5p was enriched in EVs derived from HPV- OPSCC cells. Interestingly, Honegger et al.
reported that cellular expression and packaging of miR-100-5p into EVs was increased in HeLa
cells upon silencing of cellular E6/E7 viral oncogene expression.13 Cellular expression of miR-
100-5p has previously been shown to be downregulated in response to HPV16/18 viral
infection in an E6/E7 dependent manner.11
The miRNA cargo of EVs represents a source of circulating biomarkers that may be useful in
diagnosing and monitoring cancer. Our data shows that EVs contain overlapping miRNA
profiles, but that certain miRNAs may be cancer and subtype specific. Indeed, unbiased
hierarchical clustering of EV miRNA cargo grouped samples in this study based on the HPV-
status of the producing cell. The detection of these EV-encapsulated miRNA signatures in
biological fluids might be a suitable method for surveillance of OPSCC patients to detect
catastrophic metastatic recurrence.
Acknowledgments
We would like to thank Angie Fawkes and the other members of the Edinburgh Clinical
Research Facility for their support and technical expertise with small RNA sequencing
experiments.
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Conflict of interest statement
None declared
Ethical approval
No human participants or animals were involved in this study
References
1. Chaturvedi AK, Engels EA, Pfeiffer RM, et al. Human papillomavirus and rising
oropharyngeal cancer incidence in the United States. J Clin Oncol. 2011;29(32):4294-
4301.
2. Nasman A, Attner P, Hammarstedt L, et al. Incidence of human papillomavirus (HPV)
positive tonsillar carcinoma in Stockholm, Sweden: an epidemic of viral-induced